Figure 3.

PNC formation is upregulated in LDNs. (A) LDNs expressed more CD41 in a meta-analysis of three microarray and RNA-Seq gene expression datasets. (B) Representative flow cytometry plots illustrating PNCs in LDNs. (C) LDNs have more adherent platelets compared with HDNs on flow cytometry, using EDTA as an anticoagulant. (D) LDNs from an active SLE patient were adherent to platelets. (E) ADP treatment leads to increased PNC formation compared with untreated neutrophils in whole blood (n = 7 healthy donors). (F) ADP treatment leads to increased LDNs within the PBMC fraction (n = 7 healthy donors). (G) LDNs expressed higher levels of degranulation markers such as CD63, CD15, CD66b and CD11b compared with HDNs. (H) Overlay of histograms from CD41⁻ neutrophils revealed that LDNs had increased expression of CD63, independent of platelet adhesion. Significance was determined using paired t-test and two-way analysis of variance. *P < 0.05 and ****P < 0.0001